Camptothecin (CPT) is an alkaloid extracted from Camtpotheca acuminata tree that was discovered in the 1960s (Wall et al, J. Amer. Chem. Soc. 88: 3888-3890 (1966)). This water insoluble molecule showed a very powerful anti-tumoral activity but its utilization in the clinic was limited due to very strong bladder toxicity and severe diarrhea (Gottlieb et al, Cancer Chemother. Rep. 54:461-470 (1970), Moertel et al, Cancer Chemother. Rep. 56:95-101(1972)). Structure-activity studies have identified two hydrosoluble anti-neoplastic derivatives that are currently on the market: irinotecan (CPT-11, Campto®, Camptosar®) and topotecan (Hycamtin®, a water-soluble camptothecin derivative). These two molecules are specific inhibitors of DNA topoisomerase I, that induce single strand breaks in DNA which then blocks DNA replication (Hsiang et al, J. Biol. Chem. 260:14873-14878 (1985), Kawato et al, Cancer Res. 51:4187-4191 (1991), Satoh et al, Biol. Pharm. Bull. 17:662-664 (1994)).
Irinotecan is a water soluble prodrug of SN38 (7-ethyl-10-hydroxy-camptothecin), the active metabolite which is released after hepatic enzymatic cleavage of irinotecan. SN38 is a powerful topoisomerase I inhibitor and is at least 2000-fold more active than irinotecan as an anti-proliferative agent. However, SN38 is highly water insoluble and requires delivery systems to allow its adequate administration and bioavailability. As with many camptothecin derivatives, SN38 contains a lactone ring which is highly important for anti-tumoral efficacy. This lactone ring is unstable at physiological or basic pH, resulting in the conversion of the active drug to the inactive carboxylate form (Chabot, Clin. Pharmacokinetics, 33: 245-259 (1997)).
Irinotecan is widely used for the treatment of colon cancer. However, it encounters several limitations. After administration, irinotecan has to be converted to SN38 to be active. The hepatic enzymatic conversion of irinotecan into the active drug SN38 is very partial in humans (Rohtenberg et al, J. Clin. Oncology 11: 2194-2204 (1993), Senter et al, Bioconjugate Chem. 12: 1074-1080 (2001)). Only 2 to 8% of the administered doses of irinotecan are cleaved by hepatic and tumor carboxylesterases (CES) to the lipophilic metabolite SN38 (Senter et al, Bioconjugate Chem. 12: 1074-1080 (2001), Xu et al, Clin. Cancer Res.8:2605-2611 (2002)). Analyses of the kinetics of this catabolism have demonstrated substantial inter-patient heterogeneity due to genetic and environmental factors which influence the enzyme activity by up to ten times (Charasson et al, Drug Metab. Dispos. 30:731-733 (2002)). This leads to a high level of inter-individual variability in the metabolism of irinotecan and influences the tolerance and efficacy of irinotecan and significantly complicates patient's care. (Ohe et al, J. Natl. Cancer Inst. 84:972-974 (1992), Gupta et al, Cancer Res. 54:3723-3725 (1994), Slatter et al, Drug Metab. Dispos. 28:423-433 (2000), Kraut et al, ASCO abstract No: 2501, 2004). SN38 is further converted (detoxified) to SN38-Glucuronide (SN38-G), an inactive glucurono-conjugate, in the liver by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1). Glucuronidation renders the molecule hydrophilic which permits its gastro-intestinal excretion via the bile. Once in the intestine, SN38-G is reconverted to SN38 by the intestinal bacterial flora (beta-Glucuronidase enzyme). Irinotecan itself is mainly excreted into bile (>26%) and can be converted to SN38 by intestinal CES (M. Horikawa, Pharmaceutical Res., 19: 1345-1353 (2002)). This local accumulation of SN38 in the intestine is responsible for the high level of delayed intestinal toxicity (diarrhea) observed following irinotecan treatment, which is one of irinotecan's main dose-limiting toxicities (Xie et al, Clin. Pharmacol. Ther; 72: 265-275 (2002), Alimonti et al, Cancer Treatment Rev. 30: 55-562 (2004)). The delayed diarrhea is severe (e.g., life threatening) and sometimes appears together with fever. Another significant toxicity of irinotecan is leucopenia (e.g., neutropenia). Haematological disorders may result in severe aplasia sometimes complicated by systemic infections. These severe side effects observed after treatment result in supplementary hospital care for the patients (longer hospital stay; anti-diarrhea treatment; prophylactic antibiotics therapy) (Kehrer et al, Clin. Cancer Res. 7: 1136-1141 (2001)). It has been shown in clinical trials that dose escalation/intensification of irinotecan gives an improved therapeutic response. This dose-effect has been proven in patients with colorectal metastatic cancers (Ychou et al, Cancer Chemother. Pharmacol. 50:383-391 (2002), Van Cutsem et al, Br. J. Cancer. 92:1055-1062 (2005)). However, the severe side effects described above limit the doses that can be administered to an individual, reducing irinotecan's potential efficacy.
Repeated exposition of human cancers to camptothecin derivatives can lead to the development of drug resistance (Nakagawa et al, Cancer Letters in press (2005)). This characteristic induces a decrease of efficacy not only after treatment with camptothecin derivatives but also with other commonly used anti-cancer agents.
Thus, there is a substantial interest in the development of adequate delivery systems to overcome the limitations of camptothecin derivatives (e.g. SN38), described above.
Different strategies have been proposed for delivery of camptothecin derivatives, such as liposomal formulations of SN38 (described in PCT patent application published under No WO 2004/035032 filed by NEOPHARM), nanoparticle formulations of SN38 (described in PCT patent application published under No WO 03/103596 filed by IMARX), polyglutamic acid-camptothecin conjugates (described in PCT patent application published under No WO 01/70275 filed by CELL THERAPEUTICS) or polymeric derivatives of camptothecin such as PEG-camptothecin conjugates (described in PCT patent applications published under No WO 03/097356 filed by ENZON and No WO 03/031467 filed by DEBIO) or polymeric conjugates of 20-O-[glycyl-aminoacyl-glycyl]-camptothecins (described in PCT patent application published under No WO 99/17804 filed by PHARMACIA & UPJOHN).
Peptidic drug delivery systems have also been described in PCT patent application published under No WO 00/01417 filed by CYCLACEL aiming to facilitate the delivery of different drugs, such as the 10-Hydroxycamptothecin. This patent application describes the use of homeobox peptide derived from the Drosophila antennapedia homoprotein (preferably a cell-penetrating peptide (CPP) named penetratin) for conjugation to a number of cytotoxic drugs, thus enhancing their delivery and/or therapeutic effect. However, this patent application does not show any in vitro or in vivo experiments carried out with the conjugates. Consequently the applicant has performed in vitro human serum stability studies using the conjugate described in example 29 of this patent application No WO 00/01417. These studies showed that the half life time (stability) of this conjugate is less than three minutes which does not appear sufficient for intracellular delivery of therapeutically effective amounts of the camptothecin derivative in vivo.
PCT patent application published under No. WO 01/64738 filed by DIATOS relates to amino acid sequences which react with aminoglycans and transfer a broad range of active substances (i.e. nucleic acids, proteins, drugs, antigens or antibodies) from the outside medium to the inside of cells, and more specifically cell nuclei. Such sequences derive from human proteins and are therefore non-immunogenic cell-penetrating peptides (CPP) when administered to a human in need of therapeutic treatments.
There is consequently a need in enhanced delivery efficiency, safety and efficacy of the active compound (e.g., SN38).
Within the framework of research that has lead to this invention, the applicant synthesized different CPP-camptothecin derivative conjugates. These conjugates were then evaluated in vitro and in vivo for their stability, efficacy and toxicity.
In particular, an object of the invention is to provide a compound which alleviates or decreases the drawbacks and undesired side-effects described above for camptothecin derivatives, such as for irinotecan. In particular, the present invention aims to provide a compound which is able to improve solubility of the biologically active agent in pharmaceutically acceptable forms, have sufficient stability to allow an effective intracellular delivery, reduce toxic or non-desirable side-effects, enhance the onset of action of the desired therapeutic effect, provide alternative routes for the administration of the drug, reduce inter-patient variability, and/or modify the tissue distribution and metabolism of the drug.